Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives and colorants in cosmetics to mention a few.
Because animals are unable to synthesize carotenoids de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eight C5 isoprene units (IPP). In addition, rare carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. The term “carotenoid” actually include both carotenes and xanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as “xanthophylls”. Carotenoids are furthermore described as being acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (G. Armstrong, (1999) In Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321-352).
Carotenoid biosynthesis starts with the isoprenoid pathway and the generation of a C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPP is condensed with its isomer dimethylallyl pyrophophate (DMAPP) to form the C10, geranyl pyrophosphate (GPP), and elongated to the C15, farnesyl pyrophosphate (FPP). FPP synthesis is common to both carotenogenic and non-carotenogenic bacteria. Enzymes in subsequent carotenoid pathways generate carotenoid pigments from the FPP precursor and can be divided into two categories: carotene backbone synthesis enzymes and subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase and lycopene cyclase, etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc.
Carotenoid ketolases are enzymes that introduce keto groups to the β-ionone ring of the cyclic carotenoids, such as β-carotene and zeaxanthin, to produce ketocarotenoids. Examples of ketocarotenoids include astaxanthin, canthaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, and myxobactone. Unlike genes in the upstream isoprenoid pathway that are common in many organisms, the downstream carotenoid modifying enzymes are less common.
Several classes of carotenoid ketolase have been reported (Hannibal et al., J. Bacteriol. 182: 3850-3853 (2000)). These include CrtW ketolases from Agrobacterium aurantiacum (Misawa et al., J. Bacteriol. 177(22): 6575-6584 (1995); WO 99/07867), Bradyrhizobium sp. ORS278 (Hannibal et al., supra), Brevundimonas aurantiaca (De Souza et al., WO 02/79395), Paracoccus marcusii (Yao et al., CN1380415); Bkt ketolases from Haematococcus pluvialis (Sun et al., Proc. Natl. Acad. Sci. USA, 95(19): 11482-11488 (1998); Linde, H. and Sandmann, G., EP1173579; Breitenbach et al., FEMS Microbiol. Lett., 404(2-3): 241-246 (1996)); and CrtO ketolases from Synechocystis sp. (Lagarde et al., Appl. Environ. Microbiol., 66(1): 64-72 (2000); Masamoto et al., Plant Cell Physiol., 39(5): 560-564 (2000); FR 2792335; Cheng et al., U.S. Ser. No. 10/209,372, hereby incorporated by reference)), Rhodococcus erythropolis (Cheng et al., supra), Deinococcus radiodurans (Cheng et al., supra), and Gloeobacter violaceus (Nakamura et al., DNA Res., 10: 181-201 (2003)). It should be noted that the CrtO ketolase reported in Haematococcus pluvialis (Harker, M. and Hirschberg, J., FEBS Lett., 404(2-3): 129-134 (1997); U.S. Pat. No. 5,965,795; U.S. Pat. No. 5,916,791; and U.S. Pat. No. 6,218,599) appears to be a CrtW/Bkt ketolasd based on its size and homology to other CrtW/Bkt ketolases. Sequence comparison between the Bkt ketolase from Haematococcus pluvialis to publicly available sequences mostly closely matched to other CrtW ketolases. Bkt ketolases appear to be closely related to CrtW ketolases, sharing very little structural similarity to the CrtO ketolases (Cheng, et al, supra). For example, reported CrtW/Bkt ketolases are generally encoded by nucleic acid fragments about 800-1000 bp in length while CrtO ketolases are normally encoded by a nucleic acid fragments of about 1.6 kb in size. Cheng et al. defines CrtO ketolases based on the presence of six conserved motifs considered diagnostic for all CrtO ketolases. The reported CrtO ketolases from Rhodococcus erythropolis, Deinococcus radiodurans, and Synechocystis sp. PCC6803 are comprised of these diagnostic motifs (U.S. Ser. No. 10/209,372).
The CrtO ketolases reported by Cheng et al. generally exhibit much lower activity when producing ketocarotenoids (i.e. canthaxanthin) from β-carotene in comparison to the reported CrtW ketolases (see Tables 2 and 3 in U.S. Ser. No. 10/209,372). In vitro experiments using recombinantly expressed R. erythropolis AN12 CrtO ketolase showed that after 20 hours only 30% of the initial β-carotene substrate was converted into canthaxanthin (35% of the initial β-carotene was converted to echinenone with the remaining 35% remaining as β-carotene).
There is a need for CrtO carotenoid ketolases having improved activity for ketocarotenoid production. Improvements in ketocarotenoid production will enable use of CrtO ketolases for industrial production of commercially useful ketocarotenoids, such as canthaxanthin and astaxanthin. Additionally, commercially useful CrtO ketolases can be recombinantly coexpressed with one or more structurally unrelated CrtW/Bkt ketolases to increase carotenoid titer. Coexpressing divergent ketocarotenoids should improve ketocarotenoid titer without adding instability to the host expression system.
The problem to be solved therefore is to provide nucleic acid molecules encoding CrtO ketolases useful for ketocarotenoid production.